Cell culture and gradient migration assay methods and devices

Patent metadata

Field	Value
Publication number	US-9353342-B2
Application number	US-201313761130-A
Country	US
Kind code	B2
Filing date	Feb 6, 2013
Priority date	Jan 21, 2010
Publication date	May 31, 2016
Grant date	May 31, 2016

Abstract

A number of novel improved microfluidic configurations and systems and methods of manufacture and operation for a microfluidic invasion assay system.

First claim

What is claimed is:

1. A method for culturing and monitoring cells using a microfluidic cell culture system, wherein the microfluidic cell culture system comprises a cell chamber, separated from a first microfluidic flow channel by a perfusion barrier and from a second microfluidic flow channel by a second perfusion barrier on an opposite side of the cell chamber, a gravity flow inlet well, and an outlet well, the method comprising:
introducing one or more groups of cells into the cell chamber of the microfluidic cell culture system;
culturing the one or more groups of cells in the cell chamber;
continuously perfusing the cultured one or more groups of cells by supplying a media solution to the gravity flow inlet well, wherein a medium level difference between the gravity flow inlet well and outlet well creates a perfusion flow rate;
creating and controlling a first gradient in the cell chamber by supplying a first substance to the first microfluidic flow channel and a second substance to the second microfluidic flow channel, the first gradient forming within the cell chamber perpendicular to the first perfusion barrier and the second perfusion barrier;
creating and controlling a second gradient in the cell chamber, after a selected period of time, by supplying a third substance to the first microfluidic flow channel and a fourth substance to the second microfluidic flow channel, the second gradient forming within the cell chamber perpendicular to the first perfusion barrier and the second perfusion barrier;
wherein the cell chamber provides an uninterrupted optical path to the cultured one or more cells during culturing and creation and control of the gradients;
capturing a plurality of images of the cultured one or more cells in the microfluidic culture system during culturing and during control of the gradients;
using an information system to analyze the cultured one or more cells in a sequence of the plurality of images to determine migration properties of the cultured one or more cells in response to the first and second gradients.

2. The method of claim 1 , wherein one or more of the microfluidic flow channels is associated with one or more barriers that restrict cell movement while introducing substances into the cell chamber.

3. The method of claim 1 further comprising:
controlling gradient parameters to provide a steady state or varying gradient or both at different time periods.

4. The method of claim 1 further comprising:
using microfluidic control of cell culture conditions and chemoattractant gradient formation during image capture to determine precise, dynamic quantitation of cell migration in response to stimuli.

5. The method of claim 1 further comprising:
distinguishing chemotaxis towards a substance from nondirectional migration by analyzing movements in a direction perpendicular to a gradient separately from movements in a direction parallel to the gradient.

6. The method of claim 1 further comprising:
determining total migration distance and Euclidean distance with respect to gradient conditions.

7. The method of claim 1 further comprising:
determining the effects of signaling molecules, growth factors, or other substances, on migration propensities of cells in one or more of tumors, wounds, developing tissues, immune responses, and other biological systems that can be characterized with respect to active cell migration.

8. The method of claim 1 further comprising:
culturing cells in three distinct phases:
complete substance feeding;
serum starvation; and
exposure to a chemoattractant.

9. The method of claim 1 wherein
the first microfluidic channel is in communication with a first and second solution inlet well;
the second microfluidic channel is in communication with a third and fourth solution inlet well; and
the first gradient is created by supplying solutions from the first and third solution inlet wells and
the second gradient is created by supplying solutions from the second and fourth solution inlet wells.

10. The method of claim 9 , wherein complete medium is provided in the first and fourth solution inlet wells, and complete medium without chemoattractant is provided in the second and third solution inlet wells.

11. The method of claim 1 wherein the selected period of time is a sufficient time to determine cell migration or other properties in one direction of a gradient.

12. The method of claim 1 further comprising:
capturing time-lapse images during a gradient portion or culture portion and storing digital representations of the images for analysis;
wherein images are captured at desired intervals during a gradient exposure time or a culture time or both;
analyzing the images using an information system;
wherein one or more individual cells are tracked for migratory properties across a sequential series or other series of images.

13. The method of claim 12 , further comprising:
wherein desired intervals are any number between 1 minute and 48 hour intervals;
wherein individual cells comprise one or more of about 10, about 20, about 30, about 40, about 50, about 60-100, or about 100-5,000 representative cells;
wherein the series of images are any desired regular or irregular capture of images during any desired culture period.

14. The method of claim 1 further wherein one or more individual cells are monitored in response to gradients over the course of a period of between about 6 hours to about 6 days.

15. The method of claim 1 further comprising:
analyzing cell migration properties by comparing cells in stable medium to equivalent cells exposed to a nutrient or other gradient.

16. The method of claim 1 further comprising:
analyzing cell migration properties to determine when cell migration is significantly more directed, thereby indicating a response to a gradient in the culture chamber.

17. The method of claim 1 further comprising:
determining cell migration in response to a gradient by comparing cells in a constant substance and determining whether cells in the gradient are moving substantially farther away from their site of origin than cells in a constant substance state.

18. The method of claim 1 further comprising:
determining cell migration in response to a gradient by comparing a velocity of cells in a constant substance and a velocity of cells in the gradient.

19. The method of claim 1 further comprising culturing cells for a period of between 4 hours and 6 days that allows for both cell expansion and movement with images taken some period after gradient induction.

20. A method for culturing and monitoring cells using microfluidic cell culture systems comprising:
culturing cells within a cell chamber of a microfluidic cell culture system, the cell chamber comprising a first perfusion barrier in communication with a first microfluidic flow channel and a second perfusion barrier positioned opposite from the first perfusion barrier and in communication with a second microfluidic flow channel;
continuously perfusing cells with media using gravity-driven perfusion;
creating and controlling a gradient in the cell chamber by supplying a first substance to the first microfluidic flow channel and a second substance to the second microfluidic flow channel, the gradient forming within the cell chamber along a Y-axis towards the first perfusion barrier;
monitoring cells to determine their movement in response to the gradient;
detecting, for cells exposed to the gradient, movement of cells along the Y-axis toward the

Assignees

• Emd Millipore Corp

Inventors

• Hung Ju-Sung Paul
• Lee Philip J
• Cappione Iii Amedeo J

Classifications

• C12M27/18 Primary

  Flow directing inserts · CPC title

• B01L2200/0647

  Handling flowable solids, e.g. microscopic beads, cells, particles · CPC title

• C12Q1/025

  for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18) · CPC title

• B01L3/502753

  characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation · CPC title

• B01L3/502715

  characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces · CPC title